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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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protein extraction from plant
Posted by: zia (IP Hidden, New member, 9)
Date: August 7, 2005 10:39AM
Hi, I want to extract protein from seedlings and from plant's different parts. I wan to proceed by acetone precipitation but in the start i dont have nitorgen to dry the sample. In the condition may i directly grind the sample in extraction buffer and then precipitate the protein? And whats the ration between sample and buffere for grinding and extraction of proteins that in how much buffer, how much sample be grinded. during electrophoresis what marker range is batter to use. Reagdrs
Re: protein extraction from plant
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 15, 2005 03:54PM
you do not have to freeze dry the samples. you can directly grind the sample in an extraction buffer. however the extraction buffer must contain sufficient protease inhibitors (check sigma for a plant cocktail) to prevent protein degradation and you must grind the seedlings fresh. i would suggest that you have at least twice to three times the buffer volume as the amount of tissue to be ground. the marker range depends on the weight of your protein. if it is of high molecular weight use a standard with several high molecular weight markers. if it is low, use one with several low. if you do not know the molecular weight of your protein, i would suggest using a broad range standard for right now.
Re: protein extraction from plant
Posted by: zia (IP Hidden, New member, 9)
Date: August 17, 2005 02:15AM
Thanks Respected Femmeauburn for kind suggestions. I am handling my samples according to your advise. I am using protease inhibitor 10uM in the sample (0.5g fresh material + 1.0ml extraction buffer), is it sufficient beacuse in different research papers different concentration is reported. This time i am also fcing a problem that for protein precipitation i was using 0.07% mercaptoethanol in Cold acetone. But a high amount of protein remains in the supernantent, whats the reasons may be? and what i do for this. Regards zia
Re: protein extraction from plant
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 17, 2005 11:49AM
the protein is not precipitating correctly. i've had this happen before too. one of three thing may be happening: 1). the acetone is not cold enough, 2). there is an insufficient volume of acetone used for preciptation, 3). the samples are not incubated long enough for precipitation to occur completely. i would reserve the supernatant and add additional acetone to see if i could precipitate more protein. if you can, this shows the volume of acetone you are using is not sufficient. after incubating the supernanant in acetone, centrifuge the samples as normal to pellet protein. retest the supernantant once more for protein before discarding it.
Re: protein extraction from plant
Posted by: Jacob julianah (IP Hidden, Unregistered user, )
Date: August 24, 2005 12:06PM
hi want u to hepl me on the followigng topics:
1. Composition of plant proteins 2. How is plant protins important to human 3. Apllication of plant proteins 4. What is the different between plnat proteins 5. Whhat are the common examples of plnat proteins
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